Translocation of bilirubin from blood to bile occurs in several steps, including: initial entry into the liver cell; binding to specific intrahepatocytic macromolecules; conjugation; and biliary excretion. Similar, if not identical pathways are utilized in the excretion of dyes (e.g. sulfobromophthalein (BSP), cholecystographic agents, drugs and other xenobiotics. The excretory process for such organic anions represents, therefore, a major hepatic function which is vital to survival, and often impaired hepatic injury. Nevertheless, the physiology of each of the steps in hepatic bilirubin transport is, at best, incompletely understood. This proposal seeks to clarify several aspects of hepatic bilirubin transport. Specifically, our studies will investigate: 1) The possible role of liver cell plasma membranes in organic anion transport by studying the binding of BSP and bilirubin to crude rat liver plasma membrane fractions and by isolating, purifying and characterizing the proteins responsible for binding; 2) The mechanism by which phenobarbital accelerates net hepatic bilirubin clearance (CBR) A) in rats, by comparing the effect of various doses (O-120 mg/kg/day) on CBR in vivo with their effects on the individual steps in bilirubin transport assayed in vitro, and B) in man, by assessing the effects on CBR and on the hepatic component of "early labeled" bilirubin synthesis of 1.5-2.0 mg/kg/day, a dose sufficient to increase CBR significantly, without apparent induction of bilirubin UDP-glucuronyl transferase; 3) The effect of varying exogenous and endogenous loads of bilirubin on CBR in normal man, patients with hereditary spherocytosis, and patients with Gilbert's syndrome and their parents; 4) The validity of a previously proposed compartmental model of unconjugated bilirubin metabolism by comparing model derived estimates of hepatic transport parameters in the rat with direct in vitro or in vivo measurements of the corresponding variables; and 5) The metabolism of conjugated bilirubin in the rat, in normal man, and in patients with conjugated hyperbilirubinemia, by computer analysis of plasma disappearance curves of pure radiolabeled bilirubin diglucuronide, which has recently been purified in our laboratory.